Research was conducted to investigate the biosynthesis of pertussis toxin (PT) as well as the structure and mechanism of action of the toxin. We have initiated a study to address the question of whether vaccination can provide selective pressure for antigenic variation of PT and, if so, whether antigenic drift might result in decreased efficacy of acellular pertussis vaccine. We will compare the sequence of the PT (ptx) genes of Japanese isolates of Bordetella pertussis obtained both before and after wide-spread use of acellular pertussis vaccines. In addition, we will examine whether antigenic variation of PT can affect the ability of vaccine to protect against B. pertussis. In order to do this, we will clone the PT genes from B. bronchiseptica, and exchange these genes for those found in B. pertussis. We will then examine the ability of sera from mice immunized with PT originating from B. pertussis to neutralize the bronchiseptica toxin. To date, we have obtained clinical isolates that were obtained between 1975 and the present. We have begun to clone the genes from several of the isolates obtained in the 1970s and several of the recent isolates so that we can then obtain sequence information on the ptx genes. In addition, we have cloned the ptx genes from B. bronchiseptica and are in the process of replacing the ptx genes of the bronchiseptica genes. We are continuing our work on the analysis of the secretion of PT from B. pertussis. Previously, we identified nine ptl genes that are essential for the secretaion of the toxin. In order to gain further insight into the mechanisms of secretaion, we studied PtlC which contains a putative nucleotide-binding region. We found that PtlC is cricial for secretion. Mutations in the putative nucleotide-binding region of this protein exhibit a dominant negative phenotype suggesting that PtlC may be an ATPase that provides the energy or a kinase that signals the opening of a gate or channel. Furthermore, the dominant negative phenotype suggests that PtlC interacts either with itself or other critical components of the transport apparatus. We have further investigated the architecture of the Ptl transport apparatus. Previously, we showed that PtlF interacts with Pt1I. We have extended our knowledge of the morphology of the transport structure by determining that PtlF is capable of interacting with another PtlF molecule. This work increases our knowledge of the secretion of pertussis toxin and may aid in the construction of strains of B. pertussis which efficiently produce and secrete PT. Such strains would be useful for vaccine production. In addition, knowledge of the mechanism of secretion of this toxin might aid in the development of live-attenuated vaccines that secrete inactivated forms of pertussis toxin, thus inducing a productive immune response.